An induced mRNA secondary structure enhances repZ translation in plasmid ColIb-P9

Abstract

Translation of the repZ gene encoding a DNA replication initiation protein of plasmid ColIb-P9 depends on not only the translation of a transcribed leader sequence (repY) but also the specific intergenic base pairing within RepZ mRNA between two short complementary sequences located in the repY and inc gene regions. In addition, repZ translation can be negatively regulated by Inc RNA, the product of the inc gene and a countertranscript to RepZ mRNA. Here we present evidence indicating that a stable secondary structure of RepZ mRNA, designated as structure III, sequesters one of the complementary sequences and the ribosome-binding site, thereby preventing repZ translation. When site-directed mutagenesis was used to destabilize structure III without changing the ribosome-binding site, a significant level of repZ expression was observed even in the absence of repY translation. Under these conditions, however, repZ expression could be substantially reduced by additional mutations that directly diminished the intergenic base pairing at the mRNA level between the two complementary sequences. These results indicate that repY translation is essential for the disruption of structure III by inducing the formation of a new secondary structure through the intergenic base pairing, and more importantly, that this new structure enhances repZ translation. We also found that the site of repY translation termination played a critical role in the intramolecular conformational alteration of structure III.